Using pha-1 rescue for Biolistic Transformation


as some of you will have seen from the last couple of posts, I am setting up biolistic transformation in my lab. Never one to go strictly according to the book, I am considering using a pha-1 mutant rescue as the basis for my transformation. I have a few questions regarding this idea.

  1. Although I have found references to using this approach, they are few and far between and I get the feeling also from posts to this forum that it is not a method of choice? Are there specific reasons that I have overlooked…is it a question of ease of phenotype scoring e.g. unc-119 rescue vs. pha-1 rescue, a question of the vectors available or just that other methods have taken precedence since the pha-1 approach became available?

  2. In terms of transforming the pha-1 strain, I could go the way of bombarding two separate plasmids (rescue + promoter-gene of interest) or one combined (everything in pC1 or pBX), together (or in subsequent experiments) with single-stranded oligos to promote integration.

Are the issues relating to loss of one non-integrated plasmid but not the other or the difficulty in integrating two different plasmids in the same animal the main ones to consider here?

  1. I had thought about using gfp to mark the transformed animals by creating a translational reporter. One consideration however, is that although I would include the necessary 5’ and 3’ sequences (e.g. in the genomic fragment), joining these sequences (which will mainly be coding for receptors) to gfp might create a falsely targeted or inherently unstable protein.

Does anyone have any basic tips for minimisíng the chances that the gfp tag influences placement of the receptor protein. Of course, I can check for the presence of the construct using PCR, but not where it is expressed…



I have not tried pha-1 rescue for bombardment mediated transformation, but one theoretical disadvantage is the frequency at which pha-1 mutant strains pick up spontaneous suppressors. This could cause real problems since bombardment usually involves growth of large populations and screening for rare rescued animals. We have done hundreds of unc-119 rescued bombardments and have not seen any evidence of spontaneous suppressors.

Genetics. 1991 Sep;129(1):69-77.
Suppressors of the organ-specific differentiation gene pha-1 of Caenorhabditis elegans.
Schnabel H, Bauer G, Schnabel R.


the e2123 allele of pha-1 (strain GE24) is certainly not the strongest but also not the weakest either. From the 1991 paper, the data (Table 2) doesn’t look that bad in terms of spontaneous e2123 revertants, but of course it’s a important consideration. Thanks.